ISOLATION OF A NOVEL CDNA THAT ENCODES A PROTEIN LOCALIZED TO THE PRE-ACROSOME REGION OF SPERMATIDS

We have identified a novel cDNA clone, named AZ1, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transient ly exposed to 5-azacytidine, a potent demethylating reagent. The amoun t of transcript increased with 5-azacytidine treatment of C3H10T1/2 ce lls and the transcript was highly expressed in mouse testis. As the mu tant mouse jsd/jsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed sp ecifically in spermatocytes. The mRNA was detected at significant leve ls in the testes from mice aged 16 days after birth, suggesting that i ts expression started at the pachytene spermatocyte stage. The elucida ted nucleotide sequence contained a 2841-nucleotide open reading frame , and the expected amino acid sequence had a molecular mass of 107 254 Da. Specific antibodies raised against the fusion protein including g lutathione S-transferase revealed an approximately 130-kDa band of a t ranslation product in testis and in cultured cells transfected with AZ 1 cDNA in the expression vector on Western-blot analysis. The protein was localized to the pre-acrosome region of round and elongated sperma tids. However, it was not detected at a more advanced stage of spermat ids, i.e. just before their release from Sertoli cells. This protein m ay play an important role in spermatogenesis.